The modification of proteins by the reversible conjugation of proteins containing the ubiquitin domain, is an important targeting mechanism. The enzymes that catalyze all these conjugation reactions are genetically related to those used in the ubiquitin system. Similarly, the reversal of these modifications (deconjugation) is an important regulatory pathway. In the ubiquitin pathway mutation of deubiquitinating enzymes (DUBs) have been shown to alter growth regulation, development, and cellular function. Dozens of these DUBs are known and it is likely that some of these act on ubiquitin-like proteins and not on ubiquitin itself. Others are likely involved new, undiscovered pathways of targeting or proteolysis. These assumptions can be tested biochemically by characterizing the substrate specificity of selected DUBs and then confirmed genetically, as we have done with isopeptidase T. The proposed studies will examine the structure, specificity, and physiological role of the two families of DUBs; the ubiquitin C-terminal hydrolases (UCH), the ubiquitin-specific proteases (UBP). In addition, we will define and characterize the activities of these and other enzymes which reverse the modification of proteins by ubiquitin-like proteins. Aim 1. We will characterize their physiological roles of UCH enzymes. Aim 2. The x-ray structure and regulation two UBPs (mammalian homologs of Ubpl4pp and Ubp6p) will be determined. Aim 3. Selective inhibitors of DUBs will be characterized in cell-free models to identify roles for non-K48-linked polyubiquitin chains. Aim 4. Mammalian enzymes that catalyze the deconjugation of ubiquitin-like proteins will be identified, cloned and characterized.